The invention relates to a method of preparing a crosslinked hemoglobin preparation of extended shelf life and high oxygen transport capacity, in which a stroma-free hemoglobin solution is treated with an effector and crosslinked with dialdehydes having carbon chains of 3 to 8 carbon atoms, and it relates to the hemoglobin preparation made by this method.
Efforts to use hemoglobin solutions as "blood replacement" have been made since the first half of this century. Developments began with the attempt to administer erythrocyte hemolyzates by infusion. It was found very soon, however, that the stroma components contained in these preparations had a toxic effect on the kidneys, and also affected the chemistry of coagulation. Efforts were then directed to producing stroma-free hemoglobin solutions. There were two different approaches to this. Rabiner et al. were the first to prepare a stroma-free solution in 1967 by centrifugation and ultrafiltration (Rabiner S. F. et al., J. Exp. Med., 126 1127 (1967). This method was improved by K. Bonhard (German Pat. No. 2,248,475). In recent times a crystallized, stroma-free hemoglobin was reported (De Venuto F. et al., J. Lab. Clin. Med. 89, 509 (1977). These stroma-free hemoglobin solutions, however, are not suitable for use as "blood substitute" since they have a number of decided disadvantages.
The intraerythrocytic pH is lower than that of plasma (approx. 7.2 as against 7.4). This difference in acidity causes a leftward shift of the oxygen binding curve, that is, an increase in the oxygen affinity, further intensified by withdrawal of the effector, 2,3-diphosphoglycerate (DPG), which regulates the release of oxygen in the erythrocyte. Hemoglobin solutions prepared by the described method have a half-saturation pressure (p.sub.50) of only about 16 mbar. It is possible to increase the p.sub.50 to 31 mbar in vitro by the addition of 2,3-DPG, but, in vivo, due to the weak binding of the DPG to the hemoglobin, the effector is very rapidly excreted by the kidneys. This disadvantage was eliminated by the use of a more strongly bound effector. In German Offenlegungsschrift No. 2,617,822 there is described a method using pyridoxal phosphate which leads to a hemoglobin preparation of greater oxygen yield. Although the problem of low oxygen yield has been solved, there remains a decided disadvantage in the solutions described, namely that the intravascular half life of these solutions amounts to only about 100 minutes. The hemoglobin dissolved in the plasma, due to its structure and its magnitude (molecular weight 64500 D), is rapidly eliminated by the kidneys and, if it is infused in the large amounts necessary for blood replacement, it temporarily impairs renal function.
To extend the intravascular half life a variety of methods have been used, all of them aimed at increasing the molecular weight of the hemoglobin. An attempt was made to link hemoglobin with other macromolecular polymers such as dextran (Chang, J. E., et al, Can. J. Biochem., 55, 398 (1977), hydroxyethyl starch (DE OS No. 2,616,086), gelatine (DE AS No. 2,449,885), albumin (DE AS No. 2,449,885) and polyethylene glycol (DE OS No. 3,026,398). Also, a great variety of crosslinking agents have been used for the purpose of joining the hemoglobin molecules together (DE AS No. 2,449,885, U.S. Pat. Nos. 4,001,200, 4,001,401).
All of these crosslinked preparations do have an extended intravascular half life, but they suffer from a number of other disadvantages.
Binding the hemoglobin molecules to one another or to other macromolecules by bivalent reagents does not result in products of uniform molecular weight, but rather in disperse systems of great molecular weight distribution, ranging from the four-chain basic molecule through oligomers thereof to highly polymerized molecules. What influence such a broad molecular weight distribution has on the tolerability of these solutions is not yet fully known, but it is argued that histological alterations to the kidneys and livers of experimental animals are to be attributed to polymeric and monomeric components, respectively.
Chemical modifications of the hemoglobin molecule often affect the oxygen binding curve. In most cases the affinity for oxygen is increased, so that less oxygen is yielded to tissue under physiological conditions. In other cases, the chemically altered hemoglobin receives a low charge of oxygen under the oxygen partial pressure in the lungs, so that adequate oxygen transport is not assured. The binding of effectors such as pyridoxal phosphate prior to crosslinking militates against an increase of oxygen affinity, but it has not been possible repeatably to obtain a p.sub.50 above 27 mbar at the plasmatic pH of 7.4.
The crosslinking at the same time greatly reduces the shelf life of hemoglobin solutions. The factor that limits shelf life is the formation of methemoglobin, which does not transport oxygen, and gradually renders the solution ineffectual.
It was the object of the invention to prepare a crosslinked hemoglobin preparation, that is, one of extended intravascular half life, which
(a) will have a narrow molecular weight distribution, i.e., will be free of highly polymerized hemoglobin and low in noncrosslinked hemoglobin, PA0 (b) will retain this stable molecular weight distribution and, despite the crosslinking, PA0 (c) will have a high oxygen transporting ability and PA0 (d) a good stability (shelf life). PA0 (a) by ultrafiltration; PA0 (b) by reduction with a carbonyl-group-specific reducing agent, PA0 (c) by complete chemical deoxygenation with an oxygen-consuming reducing agent, and PA0 (d) by the addition of reducing substances to the crosslinked, ultrafiltered hemoglobin preparation.